Biotechnology(Ch.20)

A Brave New World

TACGCACATTTACGTACGCGGATGCCGCGACTATGATCTACGCACATTTACGTACGCGGATGCCGCGACTATGATCACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTACATAGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCCGACTAGCATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCCTGACATCGACCTGCTCGTACATGCTAGTACATCGATCCTGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTACTAGCTACTGACTCATGATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACAATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGTTTTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGAACTCTGATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACTTCATCCGATCAGATCATGCTAGTACATCGATCGATACT

human genome3.2 billion bases

Biotechnology today• Genetic Engineering–manipulation of DNA– if you are going to engineer DNA &

genes & organisms, then you need a set of tools to work with

– this unit is a survey of those tools…

Bacteria• Bacteria review – Unicellular prokaryotes– reproduce by binary fission– rapid growth• generation every ~20

minutes• 108 (100 million) colony

overnight!– dominant form of life on

Earth– incredibly diverse

Bacterial genome

• Single circular chromosome– haploid– naked DNA• no histone proteins

–~4 million base pairs• ~4300 genes• 1/1000 DNA in eukaryote

Plasmids

• Small supplemental circles of DNA

• 5000 - 20,000 base pairs• self-replicating

– carry extra genes

• 2-30 genes • genes for antibiotic resistance

– can be exchanged between bacteria

How can plasmids help us?• A way to get genes into bacteria easily– insert new gene into plasmid– vector for gene delivery– bacteria now expresses new gene• bacteria make new protein

+

transformedbacteriagene from

other organism

plasmid

cut DNA

recombinantplasmid

vector

glue DNA

Biotechnology • Plasmids used to insert new genes into

bacteria

gene we want

cut DNA

cut plasmid DNA

insert “gene we want” into plasmid...

“glue” together

ligase

like what?…insulin…HGH…lactase

Cut DNA?DNA scissors?

recombinant plasmid

How do we cut DNA?• Restriction enzymes– restriction endonucleases– discovered in 1960s– evolved in bacteria to cut up foreign DNA • “restricted” in the sequences they cut• protection against viruses

& other bacteria

What do you notice about these phrases?

radarracecarMadam I’m AdamAble was I ere I saw Elbaa man, a plan, a canal, PanamaWas it a bar or a bat I saw?go hang a salami I’m a lasagna hog

palindromes

Restriction enzymes

• Action of enzyme – cut DNA at specific sequences• restriction site

– symmetrical “palindrome”– produces protruding ends• sticky ends• will bind to any complementary DNA

• Many different enzymes• EcoRI, HindIII, BamHI, SmaI

CTGAATTCCGGACTTAAGGC

CTG|AATTCCGGACTTAA|GGC

Discovery of restriction enzymes 1960s | 1978

Werner Arber Daniel Nathans Hamilton O. Smith

Restriction enzymes are named for the organism they come from:EcoRI = 1st restriction enzyme found in E. coli

Restriction enzymes

• Cut DNA at specific sites– leave “sticky ends”

GTAACG AATTCACGCTTCATTGCTTAA GTGCGAA

GTAACGAATTCACGCTTCATTGCTTAAGTGCGAA

restriction enzyme cut site

restriction enzyme cut site

Sticky ends

• Cut other DNA with same enzymes– leave “sticky ends” on both– can glue DNA together at “sticky ends”

GTAACG AATTCACGCTTCATTGCTTAA GTGCGAA

gene you want

GGACCTG AATTCCGGATACCTGGACTTAA GGCCTAT

chromosome want to add

gene to

GGACCTG AATTCACGCTTCCTGGACTTAA GTGCGAA

combinedDNA

Sticky ends help glue genes together

TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTTAACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA

gene you want cut sitescut sites

AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTTTTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA

chromosome want to add gene tocut sites

AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene

sticky ends

chromosome with new gene addedTAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC

CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC

sticky ends stick together

DNA ligase joins the strands Recombinant DNA molecule

Why mix genes together?

TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC

CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC

• Gene produces protein in different organism or different individual

aa aaaa aa aa aa aa aa aa aa

“new” protein from organism ex: human insulin from bacteria

human insulin gene in bacteria

bacteria human insulin

How can bacteria read human DNA?

The code is universal

• Since all living organisms… – use the same DNA– use the same code

book– read their genes

the same way

Copy (& Read) DNA• Transformation– insert recombinant plasmid

into bacteria– grow recombinant bacteria in agar cultures • bacteria make lots of copies of plasmid• “cloning” the plasmid

– production of many copies of inserted gene– production of “new” protein• transformed phenotype

DNA → RNA → protein → trait

Grow bacteria…make more

growbacteria

harvest (purify)protein

transformedbacteria

plasmid

gene fromother organism

+

recombinantplasmid

vector

A Movie, Perhaps?

QuickTime™ and aH.264 decompressor

are needed to see this picture.

Uses of genetic engineering

• Genetically modified organisms (GMO)– enabling plants to produce new proteins

• Protect crops from insects: BT corn

– corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn)

• Extend growing season: fishberries

– strawberries with an anti-freezing gene from flounder

• Improve quality of food: golden rice – rice producing vitamin A

improves nutritional value

Green with envy??Jelly fish “GFP”

Transformed vertebrates

Discovery of GFP 1960s- 1970s | 2008

Martin Chalfie Osamu Shimomura

Roger Tsien

GFP and other fluorescent proteins can be used to let us know when genes are “on” and “off”

Engineered plasmids

Selectable markerantibiotic resistance gene on plasmid

ampicillin resistanceselecting for successful transformation

successful uptake of recombinant plasmid

plasmid

ampresistance

restriction sites

EcoRI

BamHI HindIII

• Building custom plasmids– restriction enzyme sites– antibiotic resistance genes as a selectable

marker

ori

Selection for plasmid uptake

• Antibiotic becomes a selecting agent– only bacteria with the plasmid will

grow on antibiotic (ampicillin) plate

LB/amp plateLB plate

all bacteria growonly transformed

bacteria grow

a

aa a

aa

aa

aa

aaa a

a

cloning

a a

Need to screen plasmids• Need to make sure bacteria have

recombinant plasmid

plasmid

ampresistance

LacZ gene

restriction sites

lactose →blue color

recombinantplasmid

ampresistance

brokenLacZ gene

insertedgeneof interest

all in LacZ geneEcoRIBamHI

HindIII

lactose lactose →→white colorwhite colorX

origin oforigin ofreplicationreplication

Screening for recombinant plasmid

Bacteria take up plasmid Functional LacZ gene Bacteria make blue color

Bacteria take up recombinant plasmid Non-functional LacZ gene Bacteria stay white color

How could you screen for

recombinant plasmidusing pGLO

• DNA hybridization– find sequence of DNA using a labeled probe

• short, single stranded DNA molecule• complementary to part of gene of interest• labeled with radioactive P32 or fluorescent dye

– heat treat DNA in gel• unwinds (denatures) strands

– wash gel with probe• probe hybridizes with denatured DNA

Finding your gene of interest

labeled probe

genomic DNA

C T A G T C A T C

G A T C A G T A G

DNA libraries

• Cut up all of nuclear DNA from many cells of an organism– restriction enzyme

• Clone all fragments into many plasmids at same time– “shotgun” cloning

• Create a stored collection of DNA fragments– petri dish has a collection

of all DNA fragments from the organism

Making a DNA library

all DNA from many cells of an organism is cut with restriction enzymes

all DNA fragments inserted into many plasmids

engineered plasmid with selectable marker & screening system

gene of interest

clone plasmids into bacteria

1 2

3

4

?

DNA library

recombinant plasmids inserted into bacteria gene of interest

DNA Libraryplate of bacterial colonies storing & copying all genes from an organism (ex. human)

But howdo we find

colony with our gene of interest

in it?

Find your gene in DNA library• Locate Gene of Interest– to find your gene you need some of

gene’s sequence• if you know sequence of protein…–can “guess” part of DNA sequence–“back translate” protein to DNA

?

Colony BlotsCloning- plate with bacterial

colonies carrying recombinant plasmids

1

Hybridization- heat filter paper to

denature DNA- wash filter paper with

radioactive probe which will only attach to gene of interest

Replicate plate- press filter paper onto plate

to take sample of cells from every colony

3

Locate- expose film- locate colony on plate

from film

4

film

filter

plate

plate + filter

2

Problems…• Human Genome library– are there only genes in there?– nope! a lot of junk!– human genomic library has more “junk”

than genes in it

• Clean up the junk!– if you want to clone

a human gene into bacteria, you can’t have…

introns

How do you clean up the junk?

reverse transcriptase

• Don’t start with DNA…• Use mRNA– copy of the gene without the junk!

• But in the end, you need DNA to clone into plasmid…

• How do you go from RNA → DNA?– reverse transcriptase from RNA viruses• retroviruses

cDNA (copy DNA) libraries• Collection of only the

coding sequences of expressed genes– extract mRNA from

cells– reverse transcriptase • RNA →DNA • from retroviruses

– clone into plasmid• Applications– need edited DNA for

expression in bacteria• human insulin

Where do we go next….

• When a gene is turned on, it creates a trait– want to know what gene is being

expressed

proteinRNADNA trait

extract mRNA from cellsmRNA = active genes

How do you match mRNA back to DNA in cells???

reverse transcriptase

mRNA from cells

Microarrays

• Create a slide with a sample of each gene from the organism– each spot is one gene

• Convert mRNA → labeled cDNA

slide with spots of DNAeach spot = 1 gene

mRNA → cDNA

reverse transcriptase

Microarrays

• Labeled cDNA hybridizes with DNA on slide– each yellow spot = gene matched to mRNA– each yellow spot = expressed gene

slide with spots of DNAeach spot = 1 gene

cDNA matched to genomic DNAmRNA → cDNA

Application of Microarrays “DNA Chip”

• Comparing treatments or conditions = Measuring change in gene expression– sick vs. healthy; cancer vs. normal cells

– before vs. after treatment with drug

– different stages in development

• Color coding: label each condition with different color– red = gene expression in one sample

– green = gene expression in other sample

– yellow = gene expression in both samples

– black = no or low expression in both

2-color fluorescent tagging

Cut, Paste, Copy, Find…• Word processing metaphor…– cut• restriction enzymes

– paste• ligase

– copy• plasmids–bacterial transformation

• is there an easier way??

I may be very selective…

But still Ask Questions!

plasmid

ampresistance

restriction sites

EcoRI

BamHI HindIII

ori

• 1. The principal problem with inserting an unmodified mammalian gene into the bacterial chromosome, and then getting that gene expressed, is that A. prokaryotes use a different genetic code from

that of eukaryotes. B. bacteria translate polycistronic messages only. C. bacteria cannot remove eukaryotic introns. D. bacterial RNA polymerase cannot make RNA

complementary to mammalian DNA. E. bacterial DNA is not found in a membrane-

enclosed nucleus and is therefore incompatible with mammalian DNA.

2. What is the purpose of a screening gene in a plasmid?

A. To enable tranformation.B. To enable recovery of the plasmid from

solution.C. To enable identification of successful

transformants.D. To disable the spread of GE organisms

outside of the laboratoryE. To make the engineered protein product.

• 3. Which of the following is true of restriction enzymes?

A. They are capable of cutting DNA into fragments at specific points in the nucleotide sequence.

B. They form bonds between DNA fragmentsC. They are used in cell recognitionD. They are a viral defense against infection by

bacteriaE. They are found in fungi

• 4. Which of the following contains DNA from different sources?

A. Restricted DNAB. Recombinant DNAC. Reanalyzed DNAD. Reconstituated DNAE. Resurrected DNA

3. Which of the following can serve as a vector for DNA? •I. Plasmids•II. Bacteriophages•III.Animal Cells

3.I only4.II only5.III only6.I and II only7.I, II and III